This proposal is designed to improve our understanding of the molecular mechanisms involved in HIV RNA packaging by investigating the nature and specificity of HIV RNA and protein sequences that participate in packaging. During assembly, retroviruses incorporate two full length viral RNA genomes into the budding particle and this process depends upon specific but poorly defined sequences in the viral RNA and in the Gag polyprotein. A combination of in vivo and in vitro assays will be used to further investigate the molecular interactions involved in HIV RNA packaging. The first specific aim of this proposal is to identify HIV RNA sequences necessary and sufficient for packaging and to define the secondary structure features involved in specific recognition. The minimal and the optimal cis-acting packaging signal will be identified using a gene transfer assay. The structural features of this RNA segment that are important for binding to the Gag polyprotein will be elucidated using a novel and powerful in vitro genetic selection strategy. The second specific aim is to identify amino acid residues in the Gag zinc-binding domains and in other portions of the Gag precursor that contribute to specific and non-specific binding of HIV genomic RNA and the tRNA primer. Selected mutations will be introduced into a biologically active viral clone to assay their effects on particle formation, RNA packaging and viral infectivity in vivo. Some of these mutations will also be introduced into recombinant Gag proteins to assay RNA binding function in vitro. The long term goals of this research program are to increase our understanding of the HIV life cycle and to develop the information and reagents necessary for X-ray crystallography of the cis-acting packaging signal together with the appropriate segments of-the Gag polyprotein.